Lecture 18
December 4, 2012
Last time we talked about how to activate the carboxylic acid group to get the amide bond formation to
proceed under mild conditions, and in ways that will not racemize the chiral center. Just to review, some
ways to activate carbonyls are:
1. Acyl chlorides
2. p-nitrophenyl esters
3. HOBt
4. diimides (DCC, EDC, DIC)
I want to move on now to a different topic, which is how to protect the various components of amino
acids that are not reacting. In general, when you make an amide bond between two amino acids – and
you need to protect the unreacting amino group and the unreacting carboxylic acid group. Carboxylic
acid group protecting invariably uses some ester to do the protection. We often protect the nitrogen
atom with carbamates. We will talk about three of them today. For every protecting group that we
discuss, you should know how to put on the protecting group and how to remove it after the reaction is
completed.
General structure of a carbamate:
1. Cbz protecting group – carbobenzoxy – you can protect the amine by reacting it with the Cbz chloride
After the rest of the molecule (represented by the R group) is done reacting, you can take off the Cbz
group by hydrogenation –
This is a useful protecting group because the side products (carbon dioxide, toluene) are so easily
separable. CBZ is an excellent “orthogonal” protecting group for side chains on amino acids. We will talk
about what this means in a short while.
2. Boc protecting group – you protect the amine by reacting it with Boc-anhydride:
And when it’s done reacting, the Boc group is removed with trifluoroacetic acid (TFA) (relatively strong
acid):
3. F-moc protecting group – abbreviation for 9-fluoromethoxycarbonyl
The free amine is protected by reacting with Fmoc-chloride or Fmoc-OSU:
The mechanism for F-moc protection is pretty straightforward:
Nucleophilic attack of the amino group, followed by displacement of the succinimide leaving group, to
generate a protected amino acid.
Fmoc is deprotected by treating it with a secondary amine base.
The mechanism of F-moc deprotection is shown below:
The base attacks the fluorenyl proton – this proton is slightly acidic because when you form the anion,
that negative charge can be stabilized by the entire aromatic system. The anion then kicks off the
carbamate, which decomposes to lose carbon dioxide and form the desired free amine product. There
are a number of other potentially acidic protons in this molecule. The fact that the base deprotonates
only where it does is also because the anion that you form (or the quasi anion that you form during the
mechanism) is an aromatic molecule – 4n +2 with n=3.
You generally need a secondary amine to get the Fmoc deprotection to proceed in reasonable yield,
even though the mechanism for this reaction is a straightforward deprotonation. Why is the secondary
amine necessary? If you come up with a coherent explanation and send it to me by the time class starts
on Thursday, you can earn up to 5 points extra credit on the final.
Now a few practice problems: Take a sample peptide and think about how to synthesize it both
retrosynthetically and in the forward direction.
Retrosynthesis:
Forward direction:
Retrosynthetically, you should think to yourself that you need to break an amide bond – between the
nitrogen and the carbonyl group – to get back to free, unprotected amino acids as starting materials. In
this particular case, you also have an acetyl group (COMe) on the nitrogen, which does not come from
an amino acid. Our starting amino acids are proline, alanine, and lysine. The structures of each of these
three amino acids are shown below:
In the forward direction, you should remember to protect whatever functionalities are not reacting at
that particular point in time. For example, the CO2H on the starting proline is protected as a methyl
group. The nitrogen on the alanine is protected with Fmoc. Once the first coupling step is done, Fmoc is
deprotected so that it can react with the next amino acid, lysine. Lysine also has a side chain that needs
to be protected – the free NH2 group. In this case you should make sure that the lysine you introduce
has different protecting groups on the NH2 of the side chain and the NH2 of the primary amino acid
chain. This will allow you to deprotect one and leave the other intact until you are done with the whole
reaction sequence.
The idea of using a protecting group for the side chain that will not interfere with other coupling steps
and will be deprotected under mild conditions is called ORTHOGONAL PROTECTING GROUPS. You will
see the term “orthogonal” quite a bit. The best synonym I can give you is “complementary.”
After lysine is attached, you can deprotect the Boc and react it with acetyl chloride to generate the final
amide bond. TFA is used to deprotect Boc and NaOH is used to hydrolyze the ester (protecting group on
the C terminus of the peptide).
Side note: all peptides have an N terminus (where the free amino end group is) and a C terminus (where
the carboxylic acid group is). Peptides are usually grown in the C to N direction. When you try to make
these compounds, you are probably best off starting in the C direction as well.